SEARCH

SEARCH BY CITATION

Background and aims

  1. Top of page
  2. Background and aims
  3. Findings and recommendations
  4. Acknowledgements
  5. Disclosure of Conflict of Interests
  6. References
  7. Supporting Information

The central component of a blood clot is a network of fibrin fibres with which platelets and blood cells interact. The structure of this fibrin network is an important regulator of mechanical stability and resistance of the clot to fibrinolysis. Altered fibrin clot structure has consistently been reported in cardiovascular disease, including acute myocardial infarction, stable coronary artery disease, ischemic stroke, peripheral artery disease, and also venous thromboembolism [1]. Several techniques are available to determine fibrin structure, one of which characterizes the pore-size between fibrin fibres by measurement of network permeability. This method was first described by Blombäck and Okada in 1982 [2] and most laboratories currently measuring fibrin clot permeability use a variation of this method.

Permeability values (Ks) for healthy controls in previous studies range from 0.1 to 35.9 × 10−9 cm2, with a weighted average of 9.9 × 10−9 cm2. Permeability values for patients with different manifestations of atherosclerotic vascular disease or thromboembolism reported in these studies are consistently lower than those of controls and the weighted average of the difference is 2.3 × 10−9 cm2 (details in Supplementary Table S1). In addition, permeability is significantly altered in most patients with congenital or acquired dysfibrinogenemia [3–6]. Based on these studies, clot permeability in our view shows significant potential in the mechanistic characterization of arterial and venous thromboembolic diseases as well as in the elucidation of the basic science of fibrin polymerization.

However, the range of permeability values in control subjects and patients is rather large, even in comparable populations, making inter-laboratory comparison difficult. Our aim was, therefore, to perform a pilot international study to investigate whether (i) standardization of the permeability method reduces inter-laboratory variability, and (ii) lyophilized plasma can be used for standardization studies. We also characterized permeability using the standardized method in healthy Caucasian individuals (see Supplementary Figure S1 for experimental flowchart). This proposal was discussed at the 54th meeting of the Subcommittee on Factor XIII and Fibrinogen in Vienna in 2008, and the results are presented here. The data provide proof-of-concept that standardization of the clot permeability assay is feasible, that standardization of the method improves inter-laboratory variability, and that lyophilized plasma can be used as a potential future standard for the permeability assay.

Five institutions took part in the study. Each laboratory provided protocols of their currently used plasma fibrin clot permeability methods. From these a standardized protocol was developed and agreed upon by all laboratories (Supplementary Data S1). The standardized protocol was then tested in an experimental trial aimed at comparing within-day, between-day and between-laboratory variation of the standardized method with that of current in-house methods. A lyophilized pooled normal plasma sample was kindly provided and sent out by Dr Colin Longstaff, National Institute for Biological Standards and Control, Hertfordshire, UK.

Findings and recommendations

  1. Top of page
  2. Background and aims
  3. Findings and recommendations
  4. Acknowledgements
  5. Disclosure of Conflict of Interests
  6. References
  7. Supporting Information

Each laboratory analyzed the reconstituted lyophilized pool plasma using both current in-house and standardized methods. Nine replicate samples on 3 consecutive days were analyzed using in-house methods and then the procedure was repeated with the standardized method. Within- and between-laboratory variability was calculated. In addition, two laboratories also analyzed nine replicates of a locally collected fresh-frozen plasma pool, in order to compare behaviour of the lyophilized with once-frozen plasma samples.

Table 1 provides the mean ± SD permeability values and the within- and between-laboratory coefficient of variance (CV) for the lyophilized plasma. Use of a standardized permeability method moderately decreased between-laboratory variability from 29 to 24% and also decreased within- and between-day variability for each laboratory. Significant differences were, however, still present between the laboratories. This may have been the result of laboratory differences that were beyond the control of the protocol, such as precision of electronic balances, pH meters, ambient temperatures, differences in design and manufacture of the clot-holding tip, etc. In addition, operator experience appeared to be an important factor in reducing within- and between-day CVs to within acceptable levels, because technicians in laboratories 1–3 were more experienced in permeability assays than those of laboratories 4–5. Factorial ANOVA indicated that the contribution of permeability being measured in different laboratories to total variance is less for the standard than the in-house methods (F = 131 vs. F = 207). Additionally, the contribution to variability of permeability being performed in different laboratories was also much larger than the contribution of analysis being performed on different days, for both the standard and in-house methods (F = 131 vs. F = 21 and F = 207 vs. F = 56).

Table 1.   Comparison between standardized and current permeability methods using lyophilized plasma
LaboratoryDayStandardized methodCVCurrent methodsCV
Mean ± SD (× 10−9 cm2)Mean ± SD (× 10−9 cm2)
  1. *†Means with the same symbol differ between the 3 repeat days. a,b,c,dThree-day mean of standardized method, with the same letter, differs significantly from that of the current method for each laboratory. 1,2,3,4,5,6Three-day mean with the same symbol differs significantly from the 3-day mean of another laboratory for the standardized method. 1,2,3,4,5,6,7Three-day mean with the same symbol differs significantly from the 3-day mean of another laboratory for the current methods. Between-laboratory and between-day CVs were calculated combining the raw data of the individual days.

118.00 ± 0.678.411.2 ± 0.60*†5.3
28.04 ± 0.49*6.114.5 ± 1.49*10.3
37.37 ± 0.50*6.813.1 ± 0.92†7.1
3-day mean7.81 ± 0.62a,1,28.012.9 ± 1.71a13.2
219.20 ± 0.51*5.58.93 ± 0.536.0
29.03 ± 0.485.39.14 ± 0.404.4
38.52 ± 0.51*6.09.44 ± 0.293.1
3-day mean8.92 ± 0.573,46.49.20 ± 0.451,44.5
316.62 ± 0.233.58.31 ± 0.344.9
26.74 ± 0.213.08.56 ± 0.364.1
36.72 ± 0.192.98.42 ± 0.394.2
3-day mean6.69 ± 0.21b,1,3,53.18.43 ± 0.36b,2,5,64.3
415.29 ± 1.0920.53.78 ± 1.05*27.9
25.50 ± 0.427.77.41 ± 0.67*†9.0
35.73 ± 1.0718.66.00 ± 0.51†8.4
3-day mean5.52 ± 0.882,4,616.06.01 ± 1.423,4,5,723.6
5111.2 ± 1.67*14.98.25 ± 1.54*†18.7
210.8 ± 0.99†9.212.2 ± 1.56*12.8
37.33 ± 0.94*†12.811.7 ± 2.03†17.4
3-day mean9.82 ± 2.165,622.010.7 ± 2.426,722.6
Overall between laboratories7.72 ± 1.85c24.09.38 ± 2.73c29.1

To determine whether data obtained with lyophilized plasma can be extrapolated to fresh-frozen plasma (the sample material that is most commonly used in clinical studies of fibrin structure), two laboratories repeated the analysis using fresh-frozen samples from a locally collected pool of plasma from healthy individuals (Supplementary Data S2). Similar trends were observed using once-frozen plasma. Between-laboratory CV and between- and within-day CVs were decreased using the standardized assay. The permeability of clots from the lyophilized plasma pool was lower than that of once-frozen plasma clots in one laboratory [1] but not the other [5]. The permeability values for the lyophilized pool sample were, however, well within the sensitivity range of the assay and the variability was similar compared with a fresh-frozen plasma pool, indicating the suitability of lyophilized plasma as potential future standard material for this assay. Finally, we present data on the use of the standardized method of plasma clot permeability in 96 apparently healthy individuals (Supplementary Data S3).

Based on these data, we formulate the following recommendations regarding standardization of the permeability assay. (i) Standardization of the permeability assay is possible and desirable as it improves between-laboratory variation for this measurement. (ii) Operators need to be well trained in the permeability assay to reduce assay variability. (iii) While the permeability assay is predominantly used in research settings, the importance of this parameter, as a vital measurement of clot quality, justifies its extension to the clinical setting. (iv) The development of a standardized method and the application of this assay to a lyophilized sample in several laboratories in the current study demonstrate the potential for the development of an international standard based on lyophilized plasma for fibrin clot structure. In conclusion, the development of an international standard for fibrin clot structure is both feasible and of potential benefit for future clinical studies regarding the role of fibrin clot properties in cardiovascular disease and its thromboembolic manifestations.

Acknowledgements

  1. Top of page
  2. Background and aims
  3. Findings and recommendations
  4. Acknowledgements
  5. Disclosure of Conflict of Interests
  6. References
  7. Supporting Information

The authors gratefully acknowledge the technicians involved in the experimental work. We also thank the volunteers who took part in the plasma collection. We thank C. Longstaff, NIBSC, for advising on the study design, proposing the use of a standard lyophilized plasma, providing the plasma, organizing distribution of samples and suggestions on interpreting results. We express our gratitude to the Subcommittee on Factor XIII and Fibrinogen (Chaired by H.P. Kohler) of the ISTH for supporting this study.

References

  1. Top of page
  2. Background and aims
  3. Findings and recommendations
  4. Acknowledgements
  5. Disclosure of Conflict of Interests
  6. References
  7. Supporting Information
  • 1
    Undas A, Ariens RA. Fibrin clot structure and function: a role in the pathophysiology of arterial and venous thromboembolic diseases. Arterioscler Thromb Vasc Biol 2011; 12: e8899.
  • 2
    Blomback B, Okada M. Fibrin gel structure and clotting time. Thromb Res 1982; 25: 5170.
  • 3
    Sugo T, Nakamikawa C, Yoshida N, Niwa K, Sameshima M, Mimuro J, Weisel JW, Nagita A, Matsuda M. End-linked homodimers in fibrinogen Osaka VI with a B beta-chain extension lead to fragile clot structure. Blood 2000; 96: 377985.
  • 4
    Lim BC, Ariens RA, Carter AM, Weisel JW, Grant PJ. Genetic regulation of fibrin structure and function: complex gene-environment interactions may modulate vascular risk. Lancet 2003; 361: 142431.
  • 5
    Marchi R, Meyer M, De Bosch NB, Soria J, Arocha-Pinango CL, Weisel JW. Biophysical characterization of fibrinogen Caracas I with an Aalpha-chain truncation at Aalpha-466 Ser: identification of the mutation and biophysical characterization of properties of clots from plasma and purified fibrinogen. Blood Coagul Fibrinolysis 2004; 15: 28593.
  • 6
    Marchi R, Arocha-Pinango CL, Nagy H, Matsuda M, Weisel JW. The effects of additional carbohydrate in the coiled-coil region of fibrinogen on polymerization and clot structure and properties: characterization of the homozygous and heterozygous forms of fibrinogen Lima (Aalpha Arg141-->Ser with extra glycosylation). J Thromb Haemost 2004; 2: 9408.

Supporting Information

  1. Top of page
  2. Background and aims
  3. Findings and recommendations
  4. Acknowledgements
  5. Disclosure of Conflict of Interests
  6. References
  7. Supporting Information

Data S1. Protocol for the standardised permeability (Ks) method.

Data S2. Permeability standardisation experiment using once frozen plasma.

Data S3. Healthy control data.

Figure S1. Experimental flowchart of the study.

Table S1. Ks data (× 10−9 cm2) for healthy controls and CVD-related cases.

FilenameFormatSizeDescription
JTH_4883_sm_SupplementS1.docx45KSupporting info item
JTH_4883_sm_SupplementS2.docx33KSupporting info item
JTH_4883_sm_SupplementS3.docx16KSupporting info item
JTH_4883_sm_supplementS4.docx22KSupporting info item
JTH_4883_sm_SupplementS5.docx33KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.